Preparation of an infectious mononucleosis antibody neutralizing antigen and product thereof

ABSTRACT

Three antigens, termed HA, MN and FN, are prepared for the serological diagnosis of infectious mononucleosis; the HA antigen being used to identify the heterophil antibodies and the FN and MN antigens are used to differentiate them.

This is a division of application Ser. No. 689,308, filed May 24, 1976,now U.S. Pat. No. 4,139,606.

This invention relates to a serological diagnostic test for infectiousmononucleosis and to methods for preparing reagents used in that testand the method of performing the test.

For convenience and uniformity, the following designations for the testand the respective antigens have been adopted:

HAD test refers to the heterophil antibody differentiation testdescribed herein using the HA antigen prepared according to theteachings of this specification; HA refers to the heterophil antibodyreactive antigen; FN refers to the Forssman antibody neutralizingantigen; and MN refers to the infectious mononucleosis antibodyneutralizing antigen.

Infectious mononucleosis is thought to be of a viral etiology; however,it differs from the usual viral diseases in that in its course itproduces a particular type of heterophil antibody [1] in the patient'sblood. Diagnosis of infectious mononucleosis is complicated, however, bythe fact that about 4% of patients carry other classes of heterophilantibodies, such as the Forssman type antibody, serum sickness antibodyand horse sensitization antibody [1, 2, 3, 4, 7]. (Bracketed numbersrefer to literature citations at the end of the specification.)

In some respects, these antibodies are related and therefore have somesimilar reactions. But the infectious mononucleosis heterophil antibodyand the Forssman antibody, also have dissimilar properties. They reactwith substances not related to their production and each agglutinatessheep, mule or horse erythrocytes. However, they are also different. TheForssman antibodies are neutralized, according to this invention, by theFN antigen and the infectious mononucleosis antibodies are neutralizedby the MN antigen whereas the serum sickness and the horse sensitizationantibodies which are encountered rarely may be neutralized by both theFN and MN antigens. These characteristics are utilized in combinationwith new and highly advantageous antigens for the diagnosis ofinfectious mononucleosis according to the present invention.

A method, known as the Davidsohn test, [5,6] has been widely used. It isperformed by the tube method and, consequently, does not have theadvantages and characteristics of the slide diagnosis methods. In theDavidsohn test, the antigen consists of raw washed sheep erythrocytes.The differential part of the test is made with preparation from guineapig kidneys and beef erythrocytes. It has been used for a long time andhas served as a standard for many of the later tests; however, theDavidsohn test has many faults and is unsatisfactory in a number ofrespects. For example, the preparation of the sheep blood antigen istime-consuming and the resulting antigen lacks stability and uniformity;i.e., variations between individual animals. The differential part ofthe test is made by absorbing the test serum with preparations of guineapig kidneys or beef erythrocytes and involves comparative titrations ofthe absorbed and the unabsorbed serum. It is performed by the tubemethod and the results are read after several hours of reactivity.

Slide tests have been used for laboratory diagnosis of infectiousmononucleosis [8]. The antigens for such tests are prepared either fromsheep or horse erythrocytes, which are treated by various methods. Inmost cases, these antigens lack stability. Typically, about half waythrough the dated life time, the avidity of the antigen for the antibodybecomes low and the agglutinations which are formed become quite fine.This makes it difficult to read the test reactions and introduces anelement of uncertainty into the result.

A principal feature of the present invention is to provide dependablereagents with which a reliable serological diagnostic test may be madefor infectious mononucleosis.

One of the features of the invention is that the HA antigen is stronglyavid, is highly stable and is highly sensitive. It reacts with acomparatively low titer (1-8) positive infectious mononucleosis serum,producing a heavy, flocculent agglutination, which is easily read andhas a long lifetime without loss of avidity, retaining the property ofproducing a heavy, easily readable flocculent agglutination for a yearor longer, as confirmed by data summarized in Table II reportedhereinafter.

Another feature of the invention is that the MN and FN antigens are asoluble, stable type and they possess strong neutralizing properties.They are relatively clear and impart very little cloudiness to the testfield on the slide, consequently, the test remains easy to read andprovides more definitive results.

Another feature of the invention is to provide a diagnostic methodwhereby the heterophil antibody related to infectious mononucleosis maybe identified and differentiated from Forssman type (including serumsickness) antibodies in a single test; thus establishing a diagnosis ofthe disease in a few minutes time.

Another feature of the invention is to provide uniformity in testreactions during the dated time of the reagents. The antigen of thisinvention is standardized to give a distinct floccular agglutinationwith a 1-10 titer positive infectious mononucleosis serum. Through itsstability, the same type of reaction is obtained throughout a one yeardated time period and for even longer periods, as shown hereinafter inTable II. This makes it easier to read the test and makes the results ofthe test more dependable.

Another feature of the present invention resides in the composition andutilization of certain special solutions. For convenience, the solutionsare designated I-1, S-1, I-2 and S-2, the I and S designationsindicating incubation and special solutions. Typical compositions ofthese solutions are shown in Table I.

                  Table I                                                         ______________________________________                                                   Solution                                                           Component    I-1      I-2      S-1    S-2                                     ______________________________________                                        Water        2.0 l.   2.0 l.   2.0 l. 2.0 l.                                  KH.sub.2 PO.sub.4                                                                          13.6 g.  13.2 g.  19.0 gl                                                                              13.2 g.                                 K.sub.2 HPO.sub.4                                                                          --       30.0 g.  --     30.0 g.                                 NaN.sub.3    4.0 g.   4.0 g.   4.0 g. 4.0 g.                                  Tris (hydroxymethyl                                                           aminomethane)                                                                              8.0 g.   --       12.0 g.                                                                              --                                      Glucose      --       --       100.0 g.                                                                             100.0 g.                                (EDTA)K (Potassium                                                            ethylene diamine                                                              tetra-                                                                        acetic acid) --       --       --     6.0 g.*                                 ______________________________________                                         *(EDTA)K, or other anticoagulant, is added optionally for use with whole      blood in certain circumstances.                                          

The method of utilization of these solutions and the resulting antigencharacteristics are discussed in detail hereinafter. The ratios givenabove are optimum but not critical and reasonable variation in theratios and amounts of the individual constituents is comprehended withinthis invention. In general, ratio variations of from about 0.5:1 to 1:2,based on the above ratios as being 1:1, of individual constituents mayby considered generally equivalent but yield and/or quality tends todecrease with departure from the optimum ratios given in Table I.

According to this invention, heterophil antibodies in the patient'sserum, plasma or whole blood, are detected by testing the serum, plasmaor whole blood with the HA antigen prepared from horse, mule or sheepblood. The infectious mononucleosis antibody is identified byneutralization tests with the MN and FN antigens. The FN antigenneutralizes Forssman type antibodies and the MN antigen neutralizes theinfectious mononucleosis antibody. On the slide in which the antibodyhas been neutralized, the subsequent test with the HA antigen isnegative. These tests are performed on a glass slide and are visuallyread. The entire test requires only a few minutes.

According to this invention, a white-grayish HA antigen for theserological diagnosis of infectious mononucleosis is produced by theincubation of horse, mule or sheep blood in either I-1 or I-2 solutionat about pH 7 for from about 3 to 6 days at a temperature of about 36°C.

In a more particular aspect, the invention may be regarded as awhite-grayish HA antigen for the serological diagnosis of infectiousmononucleosis resulting from incubating horse, mule or sheep blood ineither I-1 or I-2 solution at about pH 7 for about 3 to about 6 days ata temperature of not more than about 36° C., separating the antigen fromblood hemoglobin and proteins, and dispersing the antigen in S-2solution for storage for a year or more, the antigen being characterizedby stability with high avidity for long storage periods of a year ormore. The antigen may be prepared from blood which has been previouslytreated with potassium ethylenediamine tetraacetic acid, or mechanicallydefibrinated.

The processes of preparing the HA antigen is an important facet of theinvention.

In another facet, the invention includes the MN antigen for use in theserological diagnosis of infectious mononucleosis prepared by heatingheavily lysed beef erythrocytes in distilled water at about 95° C. for atime of about one-half hour sufficient to extract antigen from saiderythrocytes, precipitating the MN antigen from the clear extract of thepreceding step with zinc sulphate, and separating the precipitatedantigen from the extract supernate.

The process of preparing MN antigen is also a facet of the invention.

In yet another facet, the invention includes the FN antigen for use inserological diagnosis of infectious mononucleosis prepared by suspendingground guinea pig or horse kidney tissue in saline solution; maintainingthe suspension prepared in the preceding step at a temperature of fromabout 2° to about 4° C. to thereby extract antigen into said solution;heating the suspension to about 95° C. and maintaining said temperaturefor a period of about one-half hour sufficient to extract recoverablequantities of FN antigen into the extract solution; separating thecoagulated tissue from the extract solution; adjusting the pH of theextract solution to about pH 7; precipitating the FN antigen with zincsulphate and mechanically separating the precipitated antigen from theextract solution supernate; and dispersing the antigen in S-2 solution.

The process of preparing the FN antigen is likewise an important facetof the invention.

In one important facet, the invention includes the HAD serologicaldiagnostic test for infectious mononucleosis comprising the steps ofmixing on a slide an aliquot of human blood fraction suspected tocontain the heterophil antibody characteristic of infectiousmononucleosis with a soluble MN antigen which neutralizes withoutvisible agglutination the infectious mononucleosis antibody; mixing on aslide an aliquot of said human blood fraction suspected to contain theheterophil antibody characteristic of infectious mononucleosis with asoluble FN antigen which neutralizes without visible agglutination theForssman type antibodies but not the infectious mononucleosis antibody;adding to each of the mixtures resulting from the preceding steps HAantigen prepared by the incubation of horse, mule or sheep blood in I-1or I-2 solution which antigen is agglutinative with both theunneutralized Forssman type and the unneutralized infectiousmononucleosis antibodies; and observing the results; in the case of apositive test for infectious mononucleosis, the occurrence ofagglutination on the slide having thereon the mixture containing the FNantigen and no agglutination on the slide having thereon the MN antigen.

The HA Antigen

The HA anitgen is prepared by incubating the blood of either horse, muleor sheep with an aqueous solution of potassium acid phosphate, Tris andsodium azide at pH approximately 7. The pH may suitably vary from, forexample, about 6.8 to about 7.4, and while pH 7 is optimum, the exact pHis not highly critical.

Alternatively, the aqueous incubating solution for horse, mule or sheepcomprises basic potassium phosphate, potassium acid phosphate and sodiumazide at approximately pH 7.

The blood from which the antigen is prepared may be mechanicallydefibrinated or treated with an anticoagulant such as one of the saltsof EDTA, e.g., potassium ethylenediamine tetraacetic acid (EDTA)K. Toobtain uniformity in the erythrocyte concentration, the whole blood isused and the cell volume is considered to be 40%. Preferably, thesolution contains an anticontaminant which prevents the growth ofmicroorganisms which may affect the antigen and, to this extent, acts asa preservative.

No particular concentration of Tris and/or phosphate salts is requiredbut typical concentrations are shown in the specific examples givenhereinafter. It will be understood, of course, that the specificconcentrations, steps and procedures described hereinafter are exemplaryand that considerable variation within the skill of the art may be madewithout departing from the scope, concept and teaching of the invention.

It is significant that not all systems which may be regarded as buffersare suitable for preparation of the HA antigen. In particular, it isfound that the HA antigen cannot be made using an acetate buffer.

The following example is exemplary of a suitable method for preparingthe HA antigen from horse, mule or sheep blood.

1. Thoroughly but gently mix 150 ml. of either defibrinated horse, muleor sheep blood.

2. To 2,000 ml. of distilled water add 8 gms. of Tris, 13.6 gms. ofpotassium acid phosphate and 4 gms. of sodium azide. If necessary,adjust the solution to approximately pH 7 with either Tris or withpotassium acid phosphate. This is I-1 solution.

3. Combine the blood of Step No. 1 with the solution of Step No. 2 andmix the suspension gently but thoroughly. This results in a 3%suspension, by volume, of erythrocytes based on the volume of packederythrocytes.

4. Place the suspension from Step No. 3 in a dry incubator atapproximately 36° C. and mix at about 4 hour intervals. Allow thesuspension to settle overnight and continue the process from 3 to 6days, 3 to 5 days being optimum, until a white-grayish sediment forms.Longer incubation can be used but the quality and quantity of antigen isnot increased and eventually degrades. In general the shortestincubation times which give satisfactory yield result in superiorantigen product. This white-grayish sediment is the HA antigen. The 36°C. temperature is optimum as higher temperatures tend to destroy theantigen and lower temperature extend the reaction time longer than isdesirable. Slightly lower temperatures and corresponding lengthenedincubation times may be used according to the teachings of thisinvention but with less satisfactory results.

5. After the antigen forms, from the incubation described in Step No. 4,the antigen is separated from the other blood substances, such ashemoglobin and blood proteins by centrifuging the suspension at about5,000 rpm for approximately 30 minutes. Three distinct layers formduring centrifugation, the HA antigen is a white-grayish layer and isformed as the middle of the three layers. The top and bottom layers aredevoid of antigenic activity and are discarded. The HA antigen isfurther washed with S-1 solution to remove the blood substances. In theprocess of separation, the antigen packs loosely and overlays the bottomhard-packed layer. The top layer is light and is drawn off with lowsuction. The top layer must be drawn off with considerable care to avoidloss of the antigen layer. The antigen layer is then separated from thehard-packed layer by washing it off with a syringe. The antigen is thendisbursed for a special wash with a special solution. This dispersion isconveniently done by drawing the antigen suspension into the syringe andexpressing it two or three times.

6. The antigen is then washed with special solution No. 1, referred toas S-1. S-1 is prepared by adding to 10 liters of distilled water, 500gms of glucose USP; 60 gms. of Tris; 95 gms. of potassium acidphosphate, and 20 gms of sodium azide. If necessary, the pH is adjustedto 7 with either Tris or acid potassium hydrogen phosphate.

The antigen is washed, one part antigen in twenty parts of S-1, bycentrifugation, each time separating the antigen as described in StepNo. 5. The antigen is washed until the supernate is clear and colorless.Usually, two washings are sufficient.

7. After the antigen is prepared from either horse, mule or sheep bloodand washed in the special solution S-1, it is taken out in specialsolution No. 2, S-2, which is prepared as follows. To 2 liters ofdistilled water add 13.2 gms. of potassium acid phosphate, 30 gms. ofbasic potassium phosphate, 100 gms of glucose (USP) and 4 gms. of sodiumazide. If necessary, adjust the solution to pH 7 with either the basicor the acid phosphate salt. The antigen resulting from the 150 ml. ofeither horse, mule or sheep blood is taken up in 100 ml. of S-2solution. One of the particularly unique and advantageouscharacteristics of this invention is the great flexibility permitted asto the form of the sample to be tested. Blood serum is advantageouslyused. Plasma or whole blood, however, may also be used. It is necessarythat whole blood be treated with an anticoagulant, (EDTA)K for example.If a blood sample is to be stored before use, then the anticoagulant isadded to the blood before storage. If fresh blood, from a finger or earlobe puncture for example, is the test sample, then the anticoagulant isadvantageously integrated into the antigen reagent solution. This is aparticular advantage in pediatric practice and in screening largenumbers of patients where serum separation is undesirable, impracticableor prohibitively time consuming or expensive. In the case where theanticoagulant is to be integrated into the antigen reagent, theanticoagulant, (EDTA)K for example, is conveniently formulated into thetake-up solution, S-2 solution with added (EDTA)K, in a concentration ofabout 3.0 mg/cc, being the preferred take-up solution.

The S-1 solution was found to be effective in removing hemoglobin andother blood proteins from the HA antigen without destructing itsproperties whereas the S-2 solution is effective in preserving theproperties of the HA and the other antigens referred to in this patent.

If it is desired to prepare HA antigen from EDTA treated blood, the sameprocedure is followed from Step No. 1 through Step No. 7.

If it is advantageous, the HA antigen may be prepared with the phosphatesystem, without Tris. The antigen incubation solution I-2 may beprepared, in an exemplary embodiment, as follows: To 2 liters ofdistilled water, add 13.2 gms of potassium dihydrogen phosphate, 30 gms.of potassium monohydrogen phosphate, and 4 gms. of sodium azide. Theantigen is prepared as described in Step Nos. 1 through 7 except for thesubstitution of the antigen incubation solution.

Standardization of HA Antigen

The HA antigen, which is prepared from either horse, mule or sheepblood, is standardized as follows:

1. Make serial dilution of the antigen in S-2 solution from one partantigen in one part S-2 solution to one part antigen in 15 parts S-2solution.

2. Place one drop of each dilution on a one inch square marked on aglass plate.

3. To each such drop add one drop of standard positive infectiousmononucleosis serum. Standardized human serum obtained from known casesof the disease and having a titer of 1-160.

4. Mix the reagents on each square with a wooden applicator, going fromhigh to low dilutions.

5. Tilt the plate back and forth in a rotating motion over an indirectlight and read the agglutination reaction. The dilution which reacts thefastest, and shows a heavy flocculent agglutination is the titer of theantigen. For use, the HA antigen is then made up in this dilution.

The remarkable stability of the HA antigen prepared as just described,using the S-1 wash procedure and taken up in S-2 solution, isillustrated by the date in Table II.

                  Table II                                                        ______________________________________                                                      Serum Dilution                                                  Date of Test                                                                             Lot No.  1:5   1:40 1:80 1:60 1:320                                                                              1:640                           ______________________________________                                        Start      20       4     4-   3    2    2    +1                              Start      26       4     4    3    2     2-  1                               Start + 2 mo.                                                                            20       4     3+   3    2    2    --                              Start + 2 mo.                                                                            26       4     4    3    2    2    --                              Start + 6 mo.                                                                            20       4     3    2    2    2    --                              Start + 6 mo.                                                                            26       4     4    3    2    2    --                              Start + 9 mo.                                                                            20       4     3-   2    2    2    --                              Start + 9 mo.                                                                            26       4     3+   3    2    2    --                              Start + 1 yr.                                                                            20       4     3-   2    2     2-  --                              Start + 1 yr.                                                                            26       4     3-    3-   2-   2-  --                              Start + 14 mo.                                                                           20       4     3    2    2     1+  --                              Start + 14 mo.                                                                           26       4     3    2    2     2-  --                              ______________________________________                                    

Two antigens, Lots 20 and 26, were titered, diluted 1:3 in S-2 solutionand stored in 10 cc dropper vials at 2°-4° C. The antigens were testedagainst sera prepared from a known case of infectious mononucleosishaving a titer of 1-320 in the dilutions shown in Table II. Therespective dilutions were made with S-2 solution and stored in separate10 cc vials at 2°-4° C.

MN Antigen

The MN antigen is prepared from beef erythrocytes obtained frommechanically defibrinated or (EDTA)K treated beef blood. The blood isdiluted one part of blood in one part normal saline, then the cells areseparated by centrifugation and washed twice in saline. The packed cellsare then diluted one to one in distilled water and homogenized in aWaring blender and then further diluted to a total dilution of one partof cells in four parts of distilled water. The heavily lysed cells arethen slowly heated with stirring at intervals of 5 minutes in a doubleboiler to 95° C. and kept at this temperature for a period long enoughto extract the MN antigen, about one-half hour being sufficient. Thecoagulated cells are broken up with a blender and the extract isseparated from the solid by centrifugation or filtration. The clearextract is adjusted to about pH 7 and the MN antigen is separated fromthe clear extract by precipitation with zinc sulphate and separated bycentrifugation. The procedure including the use of zinc sulphate as aprecipitating agent is another significant and important feature of thepresent invention.

A representative method for preparing MN antigen from beef blood is asfollows:

1. To 500 ml. of defibrinated beef blood, add 500 ml. of saline.

2. Separate the erythrocytes by centrifugation, and wash once more insaline.

3. Dilute the packed cells 1 to 1 with distilled water, and homogenizewith a Waring blender.

4. Further dilute the cell dispersion to a total dilution of one partcells to four parts of distilled water.

5. Heat the dispersion of cells while stirring at 5 minute intervals to95° C. and hold the dispersion at this temperature for long enough toextract the antigen, typically about thirty minutes.

6. Disperse the coagulated cells, which result from Step No. 5, in ablender and separate the extract from the solids by centrifugation orfiltration.

7. Adjust the extract to about pH 7, using, preferably, a potassiumphosphate salt.

8. Precipitate the MN antigen by adding 0.25% zinc sulphate to theextract and allow precipitate to form for one hour at room temperature.

9. Separate the precipitated antigen by centrifugation.

10. Harvest the precipitated MN antigen in 100 ml. of S-2 solution.

The MN antigen prepared as described is heat-stable and soluble.

If it is desired to use (EDTA)K treated beef blood, the same procedureis followed as described in Step Nos. 1 through 10.

Standardization of MN Antigen

MN antigen is standardized against a standard positive infectiousmononucleosis serum by determining its neutralizing effect against theantibodies of the serum. An exemplary standardization procedure is asfollows:

1. Select positive infectious mononucleosis sera which contain titers of1-800, 1-1200 and 1-1400 with HA antigen.

2. Place one drop of each serum on individual one inch squares marked ona glass plate.

3. To each square add one drop of MN antigen, mix thoroughly withseparate wooden applicators and allow to react for five minutes.

4. Add one drop of HA antigen to each dilution. Mix and observe todetermine whether or not agglutination occurs.

5. If no agglutination occurs, in all dilutions of the serum, thenadjust the MN antigen by diluting with S-2 solution, to a positiveneutralization of antibodies in a serum of 1-1200 titer. However, ifagglutination occurs in the 1-800 titer serum, then the MN antigen mustbe adjusted by concentration to neutralize a 1-1200 titer serum.

FN Antigen

The soluble FN antigen is prepared by making a saline extract of guineapig or horse kidneys, and then isolating the antigen from the extract byprecipitating with zinc sulphate. An exemplary method of preparation isas follows:

1. Cut 200 gms. of guinea pig or horse kidneys into small pieces thengrind in a Waring blender at full speed for five minutes.

2. Suspend the ground tissue in 2 liters of cooled saline, and add 4gms. of sodium azide. This results in a 10% tissue suspension and 0.2%sodium azide solution.

3. Store the 10% tissue suspension resulting from Step No. 2 at fromabout 2° to about 4° C. for about 24 hours to allow extraction.

4. Heat the suspension resulting from Step No. 3 slowly in a doubleboiler to about 95° C. and hold the heated suspension at thistemperature, with stirring, for about 30 minutes, i.e., long enough toextract recoverable amounts of FN antigen.

5. Remove the coagulated tissues, resulting from Step No. 4, bycentrifugation or filtration, and discard the sediment. The antigencontaining extract is saved and cooled to 25° C.

6. Adjust the volume of the antigen containing extract from Step No. 5with saline to a volume of 2,000 ml. and adjust to a pH of about 7 withacid or basic potassium phosphate salt.

7. Add 6 gms. (0.3%) of zinc sulphate and allow the antigen toprecipitate for about one hour at room temperature.

8. Separate the antigen by centrifugation and harvest it in 100 ml. ofS-2 solution.

The FN antigen is standardized by testing it against a standard Forssmanheterophil serum. The titer of the serum is determined by testing itwith a HA antigen. The FN antigen is then adjusted to neutralize aForssman serum having a titer of 1-400 or higher.

As described above the HAD serological diagnostic test for infectiousmononucleosis depends on the reaction of three antigens speciallyprepared according to this invention. The HA antigen readily detectsboth the infectious mononucleosis and the Forssman type heterophilantibodies. The MN antigen neutralizes the infectious mononucleosisantibody. The FN antigen neutralizes Forssman antibodies. Employingthese reactive properties of the antigens, it is possible to make anaccurate, serological diagnosis of infectious mononucleosis.

The test is performed on a presumptive and differential basis. Thespecimen is first tested with the HA antigen and, if the results arepositive, the heterophil antibodies are identified by the differentialmethod, using the FN and MN antigens.

Presumptive Test

1. Shake the HA antigen thoroughly, and place a drop of it on each oftwo one inch squares, marked on a glass plate. Then add a drop of thespecimen to the first square, and add a drop of saline to the nextsquare. Mix the reagents with separate applicators, and allow them toreact for about two minutes.

2. Read the results by slowly rotating the glass plate over an indirectlight for about two minutes. The final results should be read about fiveminutes later.

In a positive presumptive test, agglutination will occur in the firstsquare, but it will not occur in the second square. Such results showthe presence of heterophil antibodies, but, to establish a positivediagnosis for infectious mononucleosis, it is necessary to identify theantibody associated with the disease by the differential test with theFN and MN antigens. Any clumping occurring in the second square,indicates that the HA antigen is defective, and should not be used.

HAD Test

1. All the reagents must be shaken thoroughly before they are used inthe test.

2. Place a drop of either serum,* plasma, or whole blood on each ofthree one inch squares marked on a glass plate.

3. To the first square, add a drop of MN antigen; to the second square,add a drop of FN antigen; to the third square, add a drop of saline; andto each of the fourth, fifth and sixth squares, add two drops of saline.

4. Mix the reagents in the first three squares with separateapplicators, and allow them to react for about two minutes.

5. Add a drop of the HA antigen to each of the first four squares, tothe fifth square, add a drop of the MN antigen, and to the sixth square,add a drop of the FN antigen. Mix the reagents with separateapplicators, and react them for about two minutes.

6. Read the agglutination results by slowly rotating the glass plateover an indirect light for about two minutes. The final results shouldbe read five minutes later.

In a positive test, there will be no agglutination on the first square,but agglutination will occur on the second and third squares. Thefourth, fifth and sixth squares are control tests on the angigens. Ifany clumping appears on any of these squares, the respective antigen isdefective and should not be used.

These antigens become defective as a result of freezing, drying (if thevial is not stoppered properly) or insufficient dispersion.

In a negative test, there will be no agglutination in either of thesquares (indicating that there are no heterophil antibodies present), orthere will be agglutination with the MN and HA antigens, but not the FNantigen. Such reaction shows the presence of the Forssman antibody,which is neutralized by the FN antigen.

Also, neutralization may occur with both the FN and MN antigens,indicating the presence of serum sickness or horse serum sensitizationantibodies.

In rare cases, agglutination may occur in the presence of all threeantigens. This may happen if both the Forssman and the specificinfectious mononucleosis antibodies are present. It may also result ifonly one of the two antibodies is present, but its titer is higher thanthe FN or MN antigens are capable of neutralizing. A differentialtitration of the specimen must be made to resolve these conditions.

If the titer of the positive specimen is desired, it may easily beestablished by testing serial dilutions of it with the HA antigen.

The character of the agglutination formed in a positive test forinfectious mononucleosis with the HA antigen, depends on the type ofspecimen tested, and on the antibody titer of the patient's blood.Testing either serum or plasma with a high antibody titer, a heavyflocculant agglutination occurs; but if the antibody titer of thespecimen is low, then the agglutination is light and dispersed. On theother hand, if whole blood is used for the test, the type ofagglutination formed is quite different. It is stringy and resembles acobweb formation. Likewise, the agglutination varies in intensity withhigh and low antibody titers of the specimen. With a high antibodytiter, the agglutination appears in heavy shreds which tend to clump;whereas, with a low antibody titer, the shreds are light and dispersed.

Eight hundred and twenty five (825) human sera, which tested positive bythe Davidsohn presumptive method, were tested on a comparative basis bythe present invention, and the Davidsohn differential test. Thepresumptive titer of the sera varied from 1-7 to 1-896.

By the Davidsohn differential test, 674 of these sera tested positivefor infectious mononucleosis, and 151 sera tested negative. When thesame sera were tested by the method of this invention, 682 sera testedpositive and 143 sera tested negative for the disease. The results ofthe tests were consistent, in that, with both methods, the sera reactedsimilarly.

In the course of these tests, four sera were found to be of the Forssmantype.

The reliability of the whole blood method of this invention wasevaluated by comparatively testing whole blood and plasma obtained fromeighty-six (86) diagnosed cases of infectious mononucleosis. The resultsof the tests with both the whole blood and the plasma were in totalagreement, testing positive for the disease. Also, whole blood andplasma taken from sixty-five (65) healthy persons were likewise tested.Of this number, one person tested positive for the Forssman typeantibody, while the other sixth-four (64) specimens tested negative forhererophil antibodies with both the whole blood and the plasma. Inaddition, whole blood and plasma specimens were obtained from fourpeople who were known to be Forssman antibody positive, and were testedwith the antigens of this invention. Both the whole blood and the plasmatested positive for the Forssman antibody, confirming the presence ofthe Forssman antibody in these people.

Two hundred of the above sera were selected at random and comparativelytested with the antigens prepared as described in the invention fromhorse blood, mule blood and sheep blood. The horse blood antigen wasfound to be most sensitive and to possess the highest avidity with thesheep blood antigen ranking second in sensitivity and avidity and themule blood ranking third in sensitivity and avidity. All efforts toproduce antigen from donkey blood were unsuccessful.

Two hundred sera which tested negative by the sheep washed erythrocytemethod were also tested by the method of this invention and were foundto test negative.

This invention has been described by exemplary procedures, materials,and techniques to illustrate the best embodiment of the inventionpresently known to the inventor. Many alterations, modifications andvariations may, however, be made which are equivalent to the methods andprocedures and materials described in the exemplary embodiment of thespecification. Accordingly, it is intended to encompass within the scopeof this invention those methods, materials, and procedures and theequivalents thereof which fall within the broad concept of the inventionand within the spirit of the scope of the claims which are appended toand part of this specification.

References

1. Paul, J. R. and Bunnel, W. W., Am J. Med. Sci. 183:90, 1932.

2. Gailey, G. H. and Raffel, S., J. Clin. Investigation, 14:228, 1935.

3. Barrett, A. M., J. Hyg. 41:330, 1941.

4. Hoff, G. and Bauer, S., J. A.M.A., 194:351, 1965.

5. Davidsohn, I., et al, Am. J. Clin. Path., 21:1101, 1951.

6. Davidsohn, I., J. A.M.A., 108:389, 1937.

7. Forssman, J., Biochem. Z., 37:78, 1911.

8. Zichis, U.S. Pat. No. Re. 28,548.

I claim as my invention:
 1. The method of preparing an infectiousmononucleosis antibody neutralizing antigen, MN, composition comprisingheating heavily lysed beef erythrocytes dispersed in water to about 95°C. and maintaining the same at that temperature for about one-half hour,separating the solids from the mixture resulting from the precedingstep, adjusting the pH of the supernatant solution to about pH 7 withacid or basic potassium phosphate, adding zinc sulphate to the solutionto precipitate the MN antigen, separating the precipitated MN antigenfrom the supernatant solution and dispersing the antigen in a phosphatebuffer solution.
 2. The MN antigen composition prepared by the method ofclaim
 1. 3. The MN antigen composition prepared by the method of claim 1wherein the phosphate buffer comprises a solution of KH₂ PO₄, K₂ HPO₄,NaN₃ and glucose.